Molecular Mechanisms Underlying Tak1 Function in Osteoclasts

描述（由申请人提供）：骨发育受到骨形成细胞、成骨细胞和骨吸收细胞、破骨细胞的严格调控。因此，了解控制破骨细胞发生的机制对于解决骨质流失病理至关重要。破骨细胞的分化受RANK配体调控，RANK配体激活多种信号转导通路，包括MAP激酶和NF-κB通路。近端激活需要TRAF6和其他关键蛋白，包括TGF-ß活化激酶-1 （TAK1）募集到受体RANK。TRAF6、TAK1和其他信号伙伴经过广泛的翻译后修饰，旨在稳定RANK信号，并能够精确调节和执行适当的下游信号，主要是NF-κB激活。精确调控NF-κB活性对于维持正常破骨细胞活性和骨稳态至关重要。相反，这种转录因子的异常活性会导致有害的炎症性骨溶解。事实上，我们最近发现IKK2的组成激活足以在体外诱导不依赖rankl的破骨细胞生成。更令人信服的是，我们报道了组成活性IKK2的敲入导致小鼠严重的骨质流失。鉴于IKK2的磷酸化和激活是由TAK1控制的，TAK1是一种MAP激酶，与RANK-TRAF6复合物的多泛素化和稳定以及下游信号密切相关，我们决定研究其在破骨细胞发生中的分子作用。因此，我们产生了携带TAK1骨髓特异性缺失的小鼠。这些小鼠表现出骨质疏松症的所有特征，主要是破骨细胞生成缺陷。在机制上，我们观察到Tak1-null前体不能产生破骨细胞。更重要的是，我们发现包括TRAF6、NEMO和Notch-NICD在内的关键破骨细胞生成蛋白的表达减少。这种现象与NUMBL（一种先前描述过的神经元蛋白）的积累有关。与这些观察结果一致，我们在体外建立了外源表达NUMBL诱导TRAF6、NEMO、NOTCH1-NICD的降解，并抑制破骨细胞的发生。在野生型细胞中，使用优势阴性ptb -磷酸酪氨酸结合NUMBL和shRNAs抑制NUMBL可增强TRAF6和NEMO的表达，而不抑制破骨细胞的发生。此外，使用NUMBL显性阴性PTB和外源性NOTCH1-NICD表达抑制NUMBL，可以恢复tak1缺失细胞的破骨细胞生成。基于这些观察结果，我们假设“NUMBL是破骨细胞生成的抑制因子，其表达受TAK1调节。TAK1的缺失导致NUMBL蛋白的积累，从而诱导TRAF6、NEMO和NICD蛋白的降解，并随后阻断破骨细胞的发生。”为了验证这一假设，我们建议研究以下具体目标：1)确定TAK1调控NUMBL表达的机制。2)确定TAK1缺失调控和阻碍TRAF6、NEMO和NICD表达的机制。3)确定NUMBL基因消融对tak1缺失小鼠骨质疏松表型的影响。

DESCRIPTION (provided by applicant): Bone development is tightly regulated by bone forming cells, osteoblasts, and bone resorbing cells, osteoclasts. Therefore, understanding the mechanisms governing osteoclastogenesis is crucial for addressing bone loss pathologies. Differentiation of osteoclasts is governed by RANK ligand which activates several signal transduction pathways, including MAP kinases and NF-κB pathways. Proximal activation entails recruitment of TRAF6 and other key proteins including TGF-ß-activated kinase-1 (TAK1) to the receptor RANK. TRAF6, TAK1 and other signaling partners undergo extensive post-translational modifications aimed at stabilizing RANK signaling and enabling precise regulation and execution of proper down stream signals, primarily NF-κB activation. Precise regulation of NF-κB activity is crucial to maintain normal osteoclast activity and bone homeostasis. Conversely, abnormal activity of this transcription factor causes deleterious inflammatory osteolysis. In fact, we discovered recently that constitutive activation of IKK2 is sufficient to induce RANKL-independent osteoclastogenesis in vitro. More convincingly, we reported that knock-in of constitutively active IKK2 causes severe bone loss in mice. Given that IKK2 phosphorylation and activation is governed by TAK1, a MAP kinase heavily implicated in poly-ubiquitination and stabilization of RANK-TRAF6 complexes and down-stream signaling, we decided to investigate its molecular role in osteoclastogenesis. Thus, we generated mice harboring myeloid-specific deletion of TAK1. These mice displayed all hallmarks of osteopetrosis primarily defective osteoclastogenesis. Mechanistically, we observed that Tak1-null precursors fail to generate osteoclasts. More importantly, we discovered diminished expression of key osteoclastogenic proteins including TRAF6, NEMO and Notch-NICD. This phenomenon was associated with accumulation of NUMBL, a previously described neuron protein. Consistent with these observations, we established that exogenous expression of NUMBL induces degradation of TRAF6, NEMO, NOTCH1-NICD, and inhibits osteoclastogenesis in vitro. Inhibition of NUMBL using a dominant negative PTB-phosphotyrosine-binding of NUMBL and shRNAs knockdown of NUMBL enhanced expression of TRAF6 and NEMO and did not inhibit osteoclastogenesis in wild-type cells. In addition, inhibition of NUMBL using a dominant negative PTB of NUMBL and exogenous expression of NOTCH1-NICD restored osteoclastogenesis in TAK1-null cells. Based on these observations we hypothesize that "NUMBL is a repressor of osteoclastogenesis and its expression is regulated by TAK1. Deletion of TAK1 leads to accumulation of NUMBL protein which induces degradation of TRAF6, NEMO and NICD proteins, and subsequently blocks osteoclastogenesis." To test this hypothesis, we propose to investigate the following specific aims: 1) Determine the mechanism by which TAK1 regulates NUMBL expression. 2) Determine the mechanism by which TAK1 deletion regulates and impedes expression of TRAF6, NEMO and NICD. 3) Determine the effect of genetic ablation of NUMBL on the osteopetrotic phenotype of TAK1-null mice.